International Journal of

Molecular Sciences
ArticleMeasurement of Hepatic CYP3A4 and 2D6 Activity Using
Radioiodine-Labeled O-Desmethylvenlafaxine
Asuka Mizutani 1 , Masato Kobayashi 1 , Riku Aibe 2 , Yuka Muranaka 2 , Kodai Nishi 3 , Masanori Kitamura 4 ,Chie Suzuki 5 , Ryuichi Nishii 6 , Naoto Shikano 7 , Yasuhiro Magata 5 , Yasushi Ishida 8 , Munetaka Kunishima 9
and Keiichi Kawai 1,10, *
1

2

3

4

5

6

7

Citation: Mizutani, A.; Kobayashi,

8M.; Aibe, R.; Muranaka, Y.; Nishi, K.;
Kitamura, M.; Suzuki, C.; Nishii, R.;
Shikano, N.; Magata, Y.; et al.

910

Measurement of Hepatic CYP3A4
and 2D6 Activity Using RadioiodineLabeled O-Desmethylvenlafaxine.
Int. J. Mol. Sci. 2022, 23, 11458.
https://doi.org/10.3390/ijms
231911458
Academic Editors:
Giovanni Tarantino and Samuel
De Visser
Received: 24 July 2022
Accepted: 23 September 2022
Published: 28 September 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.

*

Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-0942, Ishikawa, Japan
Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, 5-11-80 Kodatsuno,
Kanazawa 920-0942, Ishikawa, Japan
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto,Nagasaki 852-8523, Nagasaki, Japan
Faculty of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho,
Matsuyama 790-8578, Ehime, Japan
Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine,
1-20-1 Handayama, Higashi, Hamamatsu 431-3192, Shizuoka, Japan
Department of Molecular Imaging and Theranostics, Institute for Quantum Medical Science, Quantum Life
and Medical Science Directorate, National Institutes for Quantum and Radiological Science and Technology,
4-9-1 Anagawa, Inage, Chiba 263-8555, Chiba, Japan
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami,
Inashiki 300-0394, Ibaraki, Japan
Department of Psychiatry, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake,Miyazaki 889-1692, Miyazaki, Japan
Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences,
Kanazawa University, Kakuma, Kanazawa 920-1192, Ishikawa, Japan
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki,
Eiheiji 910-1193, Fukui, Japan
Correspondence: kei@mhs.mp.kanazawa-u.ac.jp; Tel.: +81-76-265-2527; Fax: +81-76-234-4366

Abstract: Drug metabolizing enzyme activity is affected by various factors such as drug–druginteractions, and a method to quantify drug metabolizing enzyme activity in real time is needed. In
this study, we developed a novel radiopharmaceutical for quantitative imaging to estimate hepatic
CYP3A4 and CYP2D6 activity. Iodine-123- and 125-labeled O-desmethylvenlafaxine (123/125 I-ODV)
was obtained with high labeling and purity, and its metabolism was found to strongly involve CYP3A4
and CYP2D6. SPECT imaging in normal mice showed that the administered 123 I-ODV accumulated
early in the liver and was excreted into the gallbladder, as evaluated by time activity curves. In its
biological distribution, 125 I-ODV administered to mice accumulated early in the liver, and only themetabolite of 125 I-ODV was quickly excreted into the bile. In CYP3A4- and CYP2D6-inhibited model
mice, the accumulation in bile decreased more than in normal mice, indicating inhibition of metabolite
production. These results indicated that imaging and quantifying the accumulation of radioactive
metabolites in excretory organs will aid in determining the dosages of various drugs metabolized by
CYP3A4 and CYP2D6 for individualized medicine. Thus, 123/125 I-ODV has the potential to direct,
comprehensive detection and measurement of hepatic CYP3A4 and CYP2D6 activity by a simple andless invasive approach.
Keywords: O-desmethylvenlafaxine; whole-body imaging; CYP3A4; CYP2D6; individualized medicine

This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Drugs transit the body by four processes, absorption, distribution, metabolism, andelimination or excretion. Among these, metabolism is the process of transforming a drug

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within the body to make it more hydrophilic so that it can be excreted from the body [1–3].
In drug metabolism, drug metabolizing enzymes including cytochrome P450 (CYP) play
an essential role, and their activity differs between individuals [4], so the clinical response
to drug administration varies widely [5]. Individual specific responses to medication
can be attributed to many complex factors. These factors include the genotypes of drug
metabolizing enzymes; physiological conditions (age, sex, body size, and ethnicity); environmental influences (exposure to toxins, diet, and smoking); and pathological factors(liver and renal function, diabetes, obesity, and drug interactions), which can work alone
or in combination to influence drug responses [6]. When estimating an individual’s drug
responses, measurement of the activity and capacity of drug metabolizing enzymes isvaluable for selecting the optimal drug and determining the prescribed dose for individualized medicine. It is ideal to measure the individual drug response in real-time. Althoughgenotype analysis of CYP has been established, it only evaluates genetic polymorphisms
and is not a comprehensive analysis. There have been attempts to evaluate CYP activity
with various approaches such as blood or urine serial sampling [7,8]. However, special
chemical analysis systems, including liquid chromatography and mass spectrometry, are
not generally available in clinics or hospitals. We previously reported that whole-body
imaging with 123 I-iomazenil can directly quantify the activity of hepatic carboxylesterase bymeasuring the radioactive metabolite that accumulates in the gallbladder and urinary bladder [9]. To select radiopharmaceuticals to quantify the activity of hepatic drug-metabolizing
enzymes, important conditions are as follows: (1) the radiopharmaceutical accumulates
in a metabolic organ, (2) the radiopharmaceutical produces radioactive metabolites via
specific drug-metabolizing enzymes, (3) only the radioactive metabolite is transferred fromthe metabolic organ to an excretory organ, and (4) the amount of accumulation in theexcretory organ can be visualized and quantified. In this study, we developed a novel
radiopharmaceutical for quantitative imaging to estimate hepatic CYP3A4 and CYP2D6
activity. Among the several CYP isozymes, CYP3A4 contributes to the metabolism of
approximately 45% of all medical pharmaceuticals [10], and many prescribed drugs such as
antidepressants, antipsychotics, and antiarrhythmics are metabolized mainly by CYP3A4.
In addition, both CYP3A4 and CYP2D6 are involved in the metabolism of approximately
11% of all medical pharmaceuticals.
2. Results
2.1. Labeling of 125 I-O-Desmethylvenlafaxine
Figure 1 shows an HPLC chromatogram of iodine-125-labeled O-desmethylvenlafaxine(125 I-ODV) obtained by high-performance liquid chromatography (HPLC) with the γ-ray
detector (reaction time, 5 min; chloramine-T, 2.0 × 10−8 mol). We confirmed peak 1(retention time, 6.5–7.5 min), peak 2 (retention time, 8.5–10 min), and peak 3 (retention time,
12.5–13.5 min). To discriminate 125 I-ODV, the reaction time and the amount of chloramine-T
were changed. Figure 2 shows the percentage of peaks 1–3 by HPLC. The percentage ofpeak 3 increased while peak 1 decreased depending on the reaction time. The increased
amount of chloramine-T resulted in a similar reaction.
The increased in reaction time and chloramine-T concentration can be a factor in
the conformational change of ODV, peak 3 was estimated to be a compound in which thestructure of 125 I-ODV was changed by chloramine-T. Therefore, the peaks were identified as
peak 2 being 125 I-ODV, peak 3 being the byproduct of 125 I-ODV, and peak 1 being 125 I-NaI.
The best condition of labeling was a reaction time of 5 min and chloramine-T concentrationof 2.0 × 10−8 mol. After fractionation of 125 I-ODV, acetonitrile was removed by nitrogenrefluxing and adjusted to pH 7 with sodium hydroxide. The retention time of purified
125 I-ODV was 8.5–10 min, that of ODV (no iodinated ODV) was 4.0–4.5 min, indicating that
125 I-ODV is separated from ODV. The labeling index of 125 I-ODV was 84.5 ± 3.6%. The
radiochemical purity was more than 98% up to seven days at 4 ◦ C.

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3 of 133 of
3 of1313

125I-ODV.
Figure HPLC analysis of 125
Thechromatogram
shows
peaks
1−3.
I-ODV.The
Thechromatogram
chromatogramshows
showspeaks
peaks1−3.
1−3.
Figure 1.1.HPLC analysis of 125I-ODV.

125I-NaI.
Figure 2. The percentages of peak
peak areas
areas fromfrom thethe HPLC
HPLCchromatogram.
chromatogram. Peak
Peak 11 waswas125The
Figure 2. The percentages of peak areas from the HPLC chromatogram. Peak 1 was 125I-NaI. The
percentage ofof peakpeak 22 decreased
decreased andand thethe percentage
percentage ofof peakpeak 33 increased
increased depending
depending onon thethe reaction
reaction
percentage
percentage of peak 2 125
decreased and the percentage of peak 3 125
increased depending on the reaction
and
peak
33was
aabyproductof125125I-ODV.
time,
so peakpeak 2 waswas125125I-ODV
time,soso
I-ODV
and
peak
was
byproduct
I-ODV.
I-ODV
and
peak
3 was
a byproduct
ofof
I-ODV.
time,
peak 2 2was

2.2. Metabolismof 125inI-ODVIn Vitro
The increased
reactiontime and chloramine-T concentration can be a factor in the
The increased in reaction time and chloramine-T concentration can be a factor in the
125 I-ODV
conformationalchangeof ODV,peak
3 was estimatedto be a compoundin which
the
In
this
study,
andits
metabolite
were separated
HPLC (Figure3). With
conformational change of ODV, peak
3 was estimatedto be a bycompoundin whichthe125I-ODV was changed by chloramine-T. Therefore, the peaks were identified
structureofan NADPH-generating(NADPH[+]), an unknown
radioactive
metabolite(peak 4,structureof 125I-ODV wassystemchangedby chloramine-T.
Therefore,
the peaks
were identified
1253I-ODV
125I-ODV, peak
125
aspeak 2 time
being125
being thebyproductof125125time
I-ODV,
andmin)peakwere
1 being
retention
4–5
min)
and
(peak
5,
retention
17–18
125I-Ias peak 2 being I-ODV, peak 3 being the byproduct of I-ODV, and peak 1 beingfound,
NaI. The without
best conditionof labeling was a reaction
time of 5 minandchloramine-T
concenwhereas
the NADPH-generating
systemtime(NADPH[–]),thechloramine-T
unknown radioactive
NaI.
The best conditionof labeling was a reactionof 5 minandconcen−8 found.
trationof 2.0× not
10mol. After
fractionationof125125I-ODV,
acetonitrilewasremovedradioacby nimetabolite
was
These
results
indicatedthatthefractionoftheunknown−8
tration of 2.0 × 10 mol. After fractionation of I-ODV, acetonitrile
was removed by ni125 I-ODV.
trogenrefluxingwas
andtheadjusted
to pH 7 with metabolite
sodium hydroxide.
The retention
time of putive
metabolite
NADPH-mediated
of
With
the
presence
trogen refluxing and adjusted
to pH 7 with sodium hydroxide. The retention time of pu-of
125 I-ODV metabolite significantly increased from about 0% to 46%,
125I-ODV
NADPH
[+],
the
peak
of
rifiedwas
8.5–10min,that of ODV (no iodinated ODV) was 4.0–4.5 min, indicatrified 125I-ODV was 8.5–10
min, that of ODV (no iodinated ODV) was 4.0–4.5 min, indicat125I-ODVof
andthatthe125fraction
I-ODV significantly
from
about
85%
to 54%
ing
is125
separatedfrom ODV. decreased
The labelingindex
of 125I-ODV
was(Figure84.5 ± 4).
3.6%.
ing that I-ODV is separated from ODV. The labeling index of 125I-ODV was 84.5 ± 3.6%.
The radiochemical purity was more than 98% up to seven days at 4 °C.
The radiochemical purity was more than 98% up to seven days at 4 °C.

Int. J. Mol. Sci. 2022, 23, 11458

4,4, retention
retention time
time 4–5
4–5 min)min) andand I-ODV
I-ODV (peak(peak 5,5, retention
retention time
time 17–18
17–18 min)min) were
were found,found,
whereaswhereaswithout
withoutthe
theNADPH-generating
NADPH-generatingsystemsystem(NADPH(NADPH[–]),[–]),thetheunknown
unknownradioactive
radioactive
metabolite
metabolite waswas not
notfound.
found.These
These results
resultsindicatedindicated thatthat thethe fraction
fractionofof thethe unknown
unknown radiradi125I-ODV.
oactiveoactive metabolite
metabolite waswas thethe NADPH-mediated
NADPH-mediated metabolite
metabolite ofof 125I-ODV. With
With thethe presence
presence
125I-ODVofof NADPH
NADPH [+],[+], thethe peakpeak ofof 125I-ODV metabolite
metabolite significantly
significantly increased
increased fromfrom about
about 0%
0% to
to
125
125
4
of
13
46%,
46%, andand thethefraction
fraction ofof I-ODVI-ODV significantly
significantly decreased
decreased fromfromabout
about 85%
85%to
to54%
54%(Figure(Figure4).
4).

Figurereaction
mixtures(NADPH
Figure3.
3.HPLC
HPLCanalysis
analysisof
ofreaction
reactionmixtures
mixtures(NADPH(NADPH[[−]/[+]).
[−]/[+]).
Peak
was
not
found
forNADPH
NADPH
Figure3.
HPLCanalysisof−]/[+]).Peak
Peak444was
wasnotnotfoundfoundfor
for
NADPH
125
125
125I-ODV
125I-ODV.
dependentonthepresenceof
NADPH.
Peak
5
was
[−],
so
it
was
the
metabolite
of
125
125
I-ODV
dependentonthepresenceof
NADPH.
Peak
5
was
[−],
so
it
was
the
metaboliteof[−], so it was the metabolite of I-ODV dependent on the presence of NADPH. Peak 5 was I-ODV.
I-ODV.

125
125
125I-ODV
125I-ODV
FigureFigure4.
4.The
Thepercentage
percentageof
of125
I-ODVandmetabolite.Depending
Dependingon
onthe
thereaction
reactiontime,
time,125
Figure4.
Theand its
its metabolite.
metabolite.
Depending
on
I-ODV
125
125
125
decreased
andthemetaboliteof
I-ODV
increased.
After
30
min
of
reactiontime,thepercentage
decreased andand thethe metabolite
metaboliteofof I-ODV increased. After 30 min of reaction time, the percentage
percentageofofdecreasedof
125
125I-ODV
125
I-ODV
and
its
metabolite
becamemostlyconstant.
I-ODVand
anditsitsmetabolite
metabolitebecame
becamemostly
mostlyconstant.
constant.

125
125I-ODV.
Figure
NADPH
Figure 555 shows
shows thethe metabolite
metabolite rates
rates ofof125
I-ODV. In
In thethe absence
absence ofofNADPH
NADPH[[−][−]] and
and
FigureI-ODV.
−
andwithwithparoxetine
paroxetineandandketoconazole,
ketoconazole,thethepercentages
percentagesofof metabolite
metabolitewere
were1.52%,
1.52%,9.99%,
9.99%,and
andwithparoxetine
andketoconazole,thepercentagesofmetabolite
were
1.52%,
9.99%,
and17.6%, respectively, and the metabolite of 125 I-ODV was significantly decreased comparedwith NADPH [+].

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of 13
13
55 of

125I-ODV was significantly decreased compared
5 of 13
17.6%,respectively,
respectively,and
andthethemetabolite
metaboliteof
of125
17.6%,I-ODV was significantly decreased compared
withNADPH
NADPH[+].
[+].
with

125I-ODV. The percentage of the metabolite
Figure5.
EffectofofCYP
CYPinhibitors
inhibitorson
onthe
themetabolism
metabolismof
of125
125
FigureEffectof
I-ODV. The
The percentage
percentage ofof thethe metabolite
Figure5.5.Effect
CYP
inhibitorsonthemetabolism
of
I-ODV.
metabolite
wasdetermined
using
mouse
liver
microsomes.
With
NADPH
[−],
paroxetine,andketoconazole,
With
NADPH
[−],[−],
paroxetine,
andandketoconazole,thewas determined
determinedusing
usingmouse
mouseliver
livermicrosomes.
microsomes.
With
NADPH
paroxetine,ketoconazole,
125I-ODV significantly decreased compared with NADPH [+]. * p < 0.01 compared
themetabolite
metabolite
of
125
I-ODV
significantly
decreasedcomparedNADPH
p < compared0.01 compared
theofmetaboliteof 125
I-ODV
significantly
decreasedcompared
withwith
NADPH[+]. *[+].
p < *0.01
with
withNADPH[+].
NADPH[+].
with
NADPH[+].

123
123I-ODV
123I-ODV
123
2.3.Whole-Body
Whole-BodyImaging
Imagingof
andMetabolite
Metabolite
ininNormal
NormalMice
2.3.
Whole-Body
Imaging
ofof123
I-ODVand
Metaboliteofof123
I-ODVinNormalMice
Mice
2.3.
I-ODV
I-ODV
FigureSPECT
imagesinnormalmice.
Accumulation
is
shown
in
the
lung,
Figure666shows
showsSPECT
SPECTimages
imagesin
innormalnormalmice.
mice.Accumulation
Accumulationis
isshownshownininthe
thelung,lung,
Figureshows
liver,
andurinary
bladder(frame1).
Thoughtheaccumulation
in
the
lung
and
liver
graduliver,andurinary
bladder(frame1).
Thoughtheaccumulation
in
the
lung
and
liver
gradliver, and urinary bladder (frame 1). Though the accumulation in the lung and liver gradally
decreased,inin
theurinary
bladder
it increased(frame3). 3).
In
addition,thetheaccumulationuallydecreased,intheurinary
bladderincreased(frame3).In
Inaddition,
addition,theaccumulaually
decreased,
theurinary
bladder
ititincreased(frameaccumulamoved
tothegallbladderandintestines,
andthecontrast
between
the
liver
and
gallbladdertion moved
moved toto thethe gallbladdergallbladder andand intestines,
intestines, andand thethe contrast
contrast between
between thethe liver
liver andandtion
isgallbladderclear (frame
6).
clear(frame(frame6).
6).
gallbladderisisclear

Figure 6. Whole-body images of a normal mouse obtained at 0–10, 20–30, and 50–60 min after
Figure6.6.Whole-bodyWhole-bodyimages
imagesof
ofaanormal
normalmouse
mouseobtained
obtainedat
at0–10,0–10,20–30,
20–30,and
and50–60
50–60minminafterafter14.5
14.5
Figure123 I-ODV injection
14.5
MBq
under
1.5% isoflurane
anesthesia.
Dependingon the time,
the accu123I-ODV
MBq
injection
under
1.5%
isoflurane
anesthesia.
Depending
on
the
time,
the
accumulation
123
MBq I-ODV injection under 1.5% isoflurane anesthesia. Depending on the time, the accumulation
mulation in lung and liver decreased, and the accumulation in the gallbladder became clear. In
addition, increased accumulation in the intestines and urinary bladder indicated that excretion
was progressing.

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6 of 13
6 of 13in lung and liver decreased, and the accumulation in the gallbladder became clear. In addition, inin lung andliver decreased,
the accumulation
the gallbladderclear. In
addition,increasedaccumulationin the andintestinesand urinaryin
bladder
indicatedbecamethat excretion
wasprogress6
of13
creasedaccumulationintheintestines
andurinary
bladder
indicatedthatexcretion
was
progressing.
ing.

Figure 7 shows VOI analysis in normal mice of the liver and gallbladder. The accuFigureshows
analysisin normalof the liverwas
andgradually
gallbladder.
The accumulationin 7theliver VOI
decreased,
while
in micethegallbladderincreased.
Figure7 shows
VOI analysis
in thatnormalmiceof the liver andgallbladder.
The accumulation in the liver decreased, while that in the gallbladder was gradually increased.
mulation in the liver decreased, while that in the gallbladder was gradually increased.

Figure 7. Time activity curves in liver (a) and gallbladder (b). Mice were scanned from 0 to 60 min
Figure
7.7.Time
activity
ininlivergallbladder
(b).
Mice
were
from
123I-ODV
FigureTimeactivitycurves
curvesliver(a)(a)andgallbladder
(b).
Mice
were
scannedfrom0 was
0toto60grad60min
min
after
injection.
Theaccumulation
inand
liver
decreased,
while
that
in scanned
thegallbladder123123after
I-ODV
injection.
Theaccumulationinliver
decreased,
while
that
in
the
gallbladderwas
gradafter
I-ODV
injection.
Theaccumulationinliver
decreased,
while
that
in
the
gallbladderwasually increased.
ually
increased.
gradually
increased.

2.4. Metabolism of 125125
I-ODV in Bile of Normal and CYP-Inhibited Mice
2.4.Metabolism
Metabolismofof125I-ODV
I-ODV
Bile
Normaland
CYP-Inhibited
Mice
2.4.
in in
Bile
of of
Normaland
CYP-InhibitedMice
In the in vitro TLC study, Rf values of 125I-NaI and 125I-ODV in bile werein ranges 125of
125 I-NaI
125
125
In
the
in
vitro
thin
layer
chromatography(TLC)
study,
Rf
valuesand of IIn theandin vitro
TLC study,
Rf values
I-NaIinand
I-ODV
bileofwerein ranges
0.75–0.85
0.40–0.50,
respectively.
In ofthe bile
normaland inCYP-inhibited
mice inODV in bile
werein rangesof 0.75–0.85
andbile0.40–0.50,
respectively.
In the bile innormal
0.75–0.85
0.40–0.50,
In
theinI-ODVnormaland not
CYP-inhibited
mice
in125I-ODV,
the respectively.
fractions of 125125
I-NaI
and 125
were
found, 125
and thefracjectedwithand125 I-NaI
125I-ODV,
125I-NaI
125fractions
and CYP-inhibited
mice
injected
with
I-ODV,
the
of
and
I-ODV
were
the
fractions
of
and
I-ODV
were
not
found,
andthefracjectedwithtion of 125I-ODV metabolite (Rf value;
0.10–0.30) was confirmed (Figure 8). The fractional
125 I-ODV
125I-ODV
notof
found,
andmetabolite
the fraction
ofvalue;metabolite(Rf value; (Figure
0.10–0.30)
was fractional
confirmed
tion(Rf
0.10–0.30)
was confirmed
8). Theratio of the metabolite was more than 99%.
(Figure8).
The
fractional
ratio
of
the
metabolite
was
more
than
99%.
ratio of the metabolite was more than 99%.

Figure 8. TLC analysis radioactive metabolites in the bile of normal and CYP-inhibited mice after
125 I-ODV
125 I-NaI and
125 I-ODV
Figure
8. TLC
analysis
radioactive
metabolites
in thebile of werenormal
CYP-inhibited
mice after
injection.
The
fractions ofnotandfound
in all conditions,so theFigure8. injection.
TLC analysis
radioactive
metabolites
bile wereof normal
and CYP-inhibitedmicesoafter
125
125I-NaI andin
125the
I-ODVThe
fractions
of
I-ODV
not
found
in
all
conditions,the125of I-ODV
was selectively
excretedinI-ODVthe bile.
125metabolite
I-ODV injection.
Thewasfractionsof 125I-NaI
andin125
metaboliteof 125I-ODV
selectively
excreted
the bile. were not found in all conditions, so themetaboliteof 125I-ODV
was selectively
excreted
the bile. of 125 I-ODV in Normal and2.5. BiologicalDistributionof 125 I-ODV
and inMetabolite

CYP-Inhibited Mice
Table 1 shows the accumulation of 125 I-ODV and metabolite of 125 I-ODV in normal
mice. 125 I-ODV was rapidly distributed to organs in the whole body. The accumulation in
the lungs and kidneys significantly increased to more than 20% after 5 min of administration,and then it gradually decreased. The accumulation in the liver immediately increased
after injection and then gradually decreased, whereas the accumulation in the gallbladderInt. J. Mol. Sci. 2022, 23, 11458

7 of 13

gradually increased after injection. In the thyroid and stomach, the accumulation was low
at all time points.
Table 1. Biological distribution of 125 I-ODV and metabolite of 125 I-ODV (control).
Organ (%ID/g)

5 min

15 min30 min60 min

Blood
Brain
Heart
Lung
Thyroid *Gallbladder
Liver
SpleenPancreas
Stomach *Small intestines *Large intestines *KidneyUrine *

1.89 ± 0.03
0.80 ± 0.12
5.64 ± 0.50
27.04 ± 1.59
0.06 ± 0.01
4.31 ± 0.39
6.91 ± 1.05
9.22 ± 2.16
7.87 ± 0.60
2.44 ± 0.31
6.85 ± 1.35
1.32 ± 0.16
22.95 ± 0.43
0.63 ± 0.89

1.31 ± 0.12
0.75 ± 0.05
2.71 ± 0.18
16.75 ± 2.02
0.03 ± 0.02
5.26 ± 1.58
6.04 ± 0.94
4.79 ± 0.49
6.04 ± 0.90
3.19 ± 0.42
7.70 ± 0.85
1.18 ± 0.46
16.37 ± 0.63
3.37 ± 3.92

1.34 ± 0.29
0.73 ± 0.08
2.04 ± 0.12
13.30 ± 2.64
0.04 ± 0.00
24.23 ± 3.53
5.45 ± 1.03
3.45 ± 0.32
5.02 ± 0.73
2.75 ± 0.70
8.47 ± 0.87
0.68 ± 0.15
14.27 ± 1.20
3.69 ± 2.23

0.74 ± 0.22
0.60 ± 0.05
1.00 ± 0.24
5.01 ± 1.14
0.02 ± 0.00
33.05 ± 7.75
2.89 ± 0.61
1.75 ± 0.41
2.92 ± 0.73
1.88 ± 0.29
11.03 ± 3.03
0.47 ± 0.05
9.42 ± 2.23
7.31 ± 2.80

%ID/g means percent injected dose per gram tissue and mean ± standard deviation obtained from four mice.
* %ID/organ was calculated from %ID and measured organ weights.

Table 2 shows the accumulation of 125 I-ODV and metabolite of 125 I-ODV in CYP3A4inhibited mice. The accumulation in the liver was comparable to the control at all time
points, and the accumulation in the gallbladder was significantly lower than in the control
30 min later.
Table 2. Biological distribution of 125 I-ODV and metabolite of 125 I-ODV (CYP3A4 inhibition).
Organ (%ID/g)

5 min

15 min30 min60 min

Blood
Brain
Heart
Lung
Thyroid *Gallbladder
Liver
SpleenPancreas
Stomach *Small intestines *Large intestines *KidneyUrine *

1.91 ± 0.40
0.71 ± 0.03
5.73 ± 0.81
29.72 ± 1.47
0.05 ± 0.01
2.39 ± 1.90
5.66 ± 0.93
6.61 ± 0.70
8.93 ± 0.62 †2.37 ± 0.32
6.06 ± 0.29
1.19 ± 0.12
21.72 ± 0.85
2.10 ± 0.37

1.71 ± 0.14 ‡0.80 ± 0.09
3.93 ± 0.37 ‡21.72 ± 3.69
0.05 ± 0.01
5.43 ± 1.63
4.72 ± 1.40
6.35 ± 0.86 †5.94 ± 0.95
2.34 ± 0.53
6.37 ± 1.47
1.00 ± 0.30
19.74 ± 2.36
5.25 ± 2.28

1.44 ± 0.15
0.74 ± 0.05
2.34 ± 0.27
11.31 ± 2.60
0.09 ± 0.01 ‡9.16 ± 5.20 ‡5.03 ± 0.47
4.03 ± 0.74
23.69 ± 37.81
2.24 ± 0.34
7.71 ± 3.01
1.00 ± 0.25
15.65 ± 1.34
5.63 ± 2.03

0.65 ± 0.02
0.52 ± 0.03
0.96 ± 0.03
5.81 ± 0.35
0.11 ± 0.01 ‡20.14 ± 1.72 ‡2.12 ± 0.75
1.94 ± 0.46
2.77 ± 0.02
1.90 ± 0.37
10.07 ± 3.63
0.68 ± 0.02 ‡9.60 ± 3.05
2.01 ± 2.27

%ID/g means percent injected dose per gram tissue and mean ± standard deviation obtained from four mice.
* %ID/organ was calculated from %ID and measured organ weights. ‡ p < 0.01 and † p < 0.05 comparedwith control.

Table 3 shows the accumulation of 125 I-ODV and metabolite of 125 I-ODV in CYP2D6inhibited mice. The accumulation in the liver was comparable to the control at all time
points. In the gallbladder, the accumulation at 5 min was significantly lower than in thecontrol, while the accumulation at 15 min was significantly higher than in the control.

Int. J. Mol. Sci. 2022, 23, 11458

8 of 13

Table 3. Biological distribution of 125 I-ODV and metabolite of 125 I-ODV (CYP2D6 inhibition).
Organ (%ID/g)

5 min

15 min30 min60 min

Blood
Brain
Heart
Lung
Thyroid *Gallbladder
Liver
SpleenPancreas
Stomach *Small intestines *Large intestines *KidneyUrine *

1.83 ± 0.47
0.79 ± 0.04
5.19 ± 0.74
22.13 ± 2.64
0.05 ± 0.01
1.59 ± 1.08 ‡7.19 ± 1.14
4.62 ± 1.25 †6.02 ± 1.68
1.77 ± 0.54
7.24 ± 1.56
1.12 ± 0.16
19.94 ± 1.82
0.33 ± 0.11

1.59 ± 0.33
0.82 ± 0.18
2.87 ± 0.42
11.96 ± 0.51 †0.04 ± 0.01
10.66 ± 1.56 ‡5.56 ± 1.20
4.97 ± 0.92
5.71 ± 1.20
2.40 ± 0.38
5.64 ± 0.45
0.93 ± 0.06
17.13 ± 3.30
6.18 ± 0.48

1.12 ± 0.03
0.71 ± 0.11
1.47 ± 0.08 ‡7.77 ± 2.29 †0.05 ± 0.04
21.66 ± 2.30
5.08 ± 1.32
2.88 ± 0.59
3.94 ± 0.83
3.20 ± 0.70
10.30 ± 1.59
0.74 ± 0.25
15.90 ± 1.45
22.96 ± 2.22

0.67 ± 0.30
0.58 ± 0.18
0.62 ± 0.25
3.15 ± 0.82
0.10 ± 0.09
26.70 ± 10.19
1.75 ± 0.39 †1.46 ± 0.41
2.51 ± 0.65
2.20 ± 0.81
10.85 ± 2.01
0.67 ± 0.23
8.33 ± 2.33
33.92 ± 21.40%ID/g means percent injected dose per gram tissue and mean ± standard deviation obtained from four mice.
* %ID/organ was calculated from %ID and measured organ weights. ‡ p < 0.01 and † p < 0.05 comparedwith control.

3. Discussion
In this study, established to quantify hepatic CYP3A4 and CYP2D6 activity were
quantified using newly labeled 123/125 I-ODV.
In general, non-metabolized radiopharmaceuticals are ideal in nuclear medicine, because radiopharmaceuticals target specific functional proteins in vivo. In our previous
study, 123 I-iomazenil was metabolized by hepatic carboxylesterase, and then almost all
the radioactive metabolite moved into the gallbladder and urinary bladder [10]. We canmeasure hepatic carboxylesterase activity by detecting and quantifying the accumulationof a radioactive metabolite in the gallbladder and/or urinary bladder. Using the same
technique, here we quantified hepatic CYP activity. It is often difficult to prescribe drugs
for psychiatric pharmacotherapy in the clinic because the therapeutic index differs substantially between individuals. CYP2D6 as well as CYP3A4 contribute to the clearance ofmany clinical drugs, and many drugs are metabolized by both, including antidepressants,
antipsychotics, and analgesics. Therefore, measurement of CYP3A4 and CYP2D6 activity bya less invasive and simple approach is important for the prescription of medication. ODV isa candidate mother compound and substrate of CYP3A4 as a new radiopharmaceutical. In
the metabolic pathway of ODV, it is primarily converted to N, O-didesmethylvenlafaxine.
Hydroxylation is the main metabolic reaction of ODV, and it mainly involves CYP3A4 [11].
Although there are no reports of ODV being metabolized by CYP2D6, we expected CYP2D6
to be contributing to the metabolism of radioiodine-labeled ODV.
We were able to establish a stable radio-labeling method for ODV, and 125 I-ODV
showed very high labeling, yield, and purity (Figure 2). In NADPH-mediated metabolismof 125 I-ODV in vitro, to confirm the involvement of CYP, the effect of an NADPH-generating
system was examined. With NADPH [+], 125 I-ODV and an unknown radioactive metaboliteof 125 I-ODV were detected. In contrast, with NADPH [−], only 125 I-ODV was detected(Figure 3). Thus, an unknown radioactive metabolite was produced when the NADPHgenerating system provided energy for CYP, which was a metabolite of 125 I-ODV. To further
study the metabolism of 125 I-ODV in vitro and to clarify the specific isozyme of CYP
responsible for the metabolite of 125 I-ODV, the effects of inhibitors on the metabolism were
examined (Figure 5). Paroxetine and ketoconazole significantly inhibited the metabolismof 125 I-ODV (79% and 63% inhibition). Therefore, 125 I-ODV was mainly metabolized by
CYP3A4 and CYP2D6. Although non-labeled ODV is metabolized only by CYP3A4 [11],125 I-ODV was found to be metabolized by both CYP3A4 and CYP2D6.
On imaging after 123 I-ODV injection, the accumulation in the liver decreased, while
accumulation in the gallbladder increased over time (Figure 7). To set a VOI on SPECT
images, it is necessary that the accumulation in the targeted organ does not overlap andInt. J. Mol. Sci. 2022, 23, 11458

9 of 13is clear. In normal mice, the gallbladder/liver ratio increased over time (0.62 to 11.43,
Figure 7). Thus, 123 I-ODV was excreted into the bile, confirming that the VOI can beeasily set in the gallbladder. To confirm the radioactive materials in bile, gallbladders
were collected from 125 I-ODV-injected mice 60 min after injection. As shown in Figure 8,
metabolites of 125 I-ODV that are not 125 I− or 125 I-ODV were found to be present in bileat a rate of more than 99%. This result indicated that 125 I-ODV in the mouse liver was
metabolized, and the metabolite of 125 I-ODV was selectively excreted to the bile. Similarly,
in CYP3A4- and CYP2D6-inhibited mice, only 125 I-ODV was found in the bile. Thus, wefound that inhibition of 125 I-ODV metabolism did not result in metabolites being produced
and excreted by another metabolic pathway.
Imaging showed that the metabolite of 123 I-ODV was excreted in the bile and VOI
was more likely to be set in the gallbladder, so biological distribution was performed
for a more quantitative evaluation. The biological distribution in normal mice based on
examining each organ showed similar results to whole-body imaging. For the biologicaldistribution of 125 I-ODV and metabolite of 125 I-ODV (Table 1), the accumulation of 125 IODV and metabolite of 125 I-ODV was high in the blood, lung, and liver (a metabolic organ),in addition to the gallbladder and kidney (excretory organs), immediately after injection.
The accumulation in the liver decreased over time. By contrast, accumulation in the
gallbladder and kidney increased early on. These results indicate that 125 I-ODV is excreted
via enterohepatic and renal routes. In addition, low accumulation in the thyroid andstomach indicated that 125 I-ODV was almost never metabolized to 125 I− . Thus, 125 I-ODV
accumulated and metabolized in the liver, and the metabolite of 125 I-ODV was excreted to
the gallbladder. Since in the in vitro study, 125 I-ODV was mainly metabolized by CYP3A4
and CYP2D6, we focused on CYP3A4 and CYP2D6 in the in vivo study. In model mice with
reduced CYP3A4 and CYP2D6 activity (Tables 2 and 3), the accumulation in the liver was
similar to that in control mice at all time points. In contrast, in the model mice with reduced
CYP3A4, the accumulation in the gallbladder at 5 and 15 min after injection of 125 I-ODV
was not significantly different from that of the control. However, at 30 and 60 min, theaccumulation in the gallbladder was lower than that of control, indicating that the decrease
in metabolite production due to reduced CYP3A4 activity was confirmed as a decreasein the amount of metabolites accumulation in the bile. In the model mice with reduced
CYP2D6, the accumulation in the gallbladder was significantly lower at 5 min, higher at
15 min, and similar after 30 and 60 min compared to controls. The effect of paroxetine
on CYP2D6 inhibition differed in each mouse and may have a significant effect on thevariability of the accumulation in the gallbladder of the metabolite of 125 I-ODV. Therefore,
it is difficult to create a mouse model using CYP inhibitors to evaluate CYP activity, but
this could be improved by using CYP-deficient mice.
These results indicate that 123/125 I-ODV meets four important conditions for quantitative imaging of CYP activity: (1) 123/125 I-ODV accumulates in the liver (a metabolic organ),(2) 123/125 I-ODV is metabolized specifically by CYP3A4 and CYP2D6, and the metabolite isradioactive, (3) the metabolite of 123/125 I-ODV transfers from the liver to the gallbladder,and (4) accumulation in the excretory organs can be visualized and quantitated.
4. Materials and Method
4.1. Materials
Methanol, chloramine-T, ethylene diamide tetra acetic acid (EDTA), and glucose-6phosphate (G6P) were purchased from Nacalai tesque (Kyoto, Japan). β-Nicotinamideadenine dinucleotide phosphate (β-NADP+ ), sodium hydroxide and isoflurane werepurchased from Fujifilm Wako Pure Chemical Industries (Osaka, Japan), and glucose6-phosphate dehydrogenase (G6PD) was purchased from Oriental Yeast (Osaka, Japan).
O-Desmethylvenlafaxine was purchased Tokyo Chemical Industry (Tokyo, Japan). 125 I-NaI
was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). 123 I-NaI
was purchased from Fujifilm Toyama Chemical (Tokyo, Japan). All other reagents andchemicals were of analytical or high-performance liquid chromatography HPLC grade.

phate dehydrogenase (G6PD) was purchased from Oriental Yeast (Osaka, Japan). ODesmethylvenlafaxine was purchased Tokyo Chemical Industry (Tokyo, Japan). 125I-NaI
was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). 123I-NaI waspurchased from Fujifilm Toyama Chemical (Tokyo, Japan). All other reagents and chemicals were of analytical or high-performance liquid chromatography (HPLC) grade.
Int. J. Mol. Sci. 2022, 23, 11458

10 of 134.2. Labeling of

125

I-O-Desmethylvenlafaxine

O-Desmethylvenlafaxine (ODV) is a pharmacologically active metabolite of ven4.2.
Labelingof 125 I-ODV
lafaxine,
which
is a clinical antidepressant. ODV is mainly metabolized to N, Odidesmethylvenlafaxine
by CYP3A4
Thus, ODV
was selected
as isa asubstrateof
ODV is a pharmacologically
active[11].
metaboliteof venlafaxine,whichclinical anCYP3A4
asaparentcompound
for
a
novel
radiopharmaceuticalwiththeexpectationthattidepressant. ODV is mainly metabolized to N, O-didesmethylvenlafaxine by CYP3A4 [11].
it would
be wasa substrateforasCYP3A4.
Thus,
ODV
selecteda substrate of CYP3A4 as a parent compound for a novel
ODV was labeledbytheradioiodine(FigureA total
2.0 × 10−9formol
ODV was disradiopharmaceuticalwithexpectationthat it9).
would
be of
a substrate
CYP3A4.
−9−8
9 mol ODV wassolvedby was100 µLmethanol,and 2.0 × 10
, 2.0 ×9).
10 A
, and
10−7×molwas
ODV
labeledby radioiodine(Figuretotal2.0of× 2.0
10−chloramine-T−9 , 2.0 × were
−7MBq
125I-NaI anddissolvedby
by100
20 µL distilledwater.
These
dissolved
methanol,and 2.0× 10solutions
10−8 ,mixed
and 2.0with
× 103.7
mol chloramine125I-ODVat room
temperature
5–15Thesemin. The
separationand purificationof 125
Tincubated
was dissolved
by 20µL distilledfor
water.
solutionswere mixed
with 3.7 MBq
I-NaI
were
performed
by
an
HPLC
systemconsisting
of
a
pump
(model
L-7100,
Hitachi,Japan),and incubated at room temperature for 5–15 min. The separation and purification of 125 Ia UVwere
detector(Chromasterandofa aγ-ray
detector
ODV
performedby an 5410,
HPLCHitachi,
system Japan),consistingpump(model(model
L-7100,RLC-701,
Hitachi,Japan),a UV detector(Chromaster
5410, Hitachi,
and a γ-ray detector(model
Hitachi-Aloka
Medical,Japan) equippedwith aJapan),
5C18 AR-II-column(4.6 × 250
mm; 5RLCµm,
701,
Hitachi-Aloka
Medical, Japan)
equippedwith aJapan)
5C18 AR-II-column(4.6 processing.
× 250 mm;
Nacalaitesque). A PowerChrom(ver.
2.3.3, eDAQ,
was used for data
5The
µm,mobile
Nacalaitesque).
A PowerChrom
2.3.3,eDAQ,
for data
4 buffer(pH Japan)
4.0) andwas
30%usedacetonitrileatphase
consistedof 70% 20 mM(ver.
KH2POprocessing.
mobile phaseconsisted
70% 20mM
KH2 PO4 at
buffer(pHUV.
4.0)Theandstabil30%
flow rate ofThe
1.0 mL/min.
The elutionof 125ofI-ODV
wasmonitored
254 nm
125Iacetonitrile
at flowrateof 1.0 mL/min.
of 125 I-ODV (TLC).
was monitored
at 254ity of 125I-ODV
wasanalyzedby thin The
layerelution
chromatography
The elution
of nm
125
125
UV.
The stability
elutionof I-ODVwas
spottedODVwasspottedofontoI-ODVa silicawas
gelanalyzed
TLC plateby60TLC.
F254The(Merck,Darmstadt,
Germany).
The
ontosilicaandgel theTLCTLCplate
60 Fwere
Darmstadt,
Germany). The TLC
andthe
TLC aplatespots
developed
using methanol/acetate
at a plate
ratio of100:1.
254 (Merck,
TLC
were developed
using methanol/acetate
a ratioof 100:1.
After
Afterspots
development
and complete
drying, the TLC atplateswere
cut into
21 development
fractions, andandcompletedrying,theTLC
plates
were
cut
into
21
fractions,andthethe radiography associated with each fraction was quantified using a γ-rayradiography
counter (Acassociatedwith Aloka,
each fraction
quantified
using aratios
γ-rayofcounter
7000,
125I− and(AccuFLEXγ
125I-ODV were
cuFLEXγ 7000,
Tokyo, wasJapan).
The fractional
cal125 I− and 125 I-ODV were calculated by
Aloka,
Tokyo,
Japan).
The
fractionalratiosofculated by dividing the radioactive counts for each fraction by the total radioactivity
dividing
count. the radioactive counts for each fraction by the total radioactivity count.

125 I-labeling scheme of ODV. ODV was labeled with 125 I-NaI by oxidation reaction.
FigureFigure9.9. 125I-labelingscheme of ODV. ODV was labeled with 125I-NaI by oxidation reaction.

4.3. Metabolism of 125 I-ODV In Vitro
Animal studies were approved by the Animal Care Committee at Kanazawa University (AP-173851) and were conducted in accordance with international standards foranimal welfare and institutional guidelines. For preparation of the pooled mouse liverhomogenates (MLH), three six-week-old ddY mice (Japan SLC, Tokyo, Japan) were euthanized under anesthesia with isoflurane, and the removed livers were weighed. Afteradding Krebs–Ringer phosphate buffer (pH 7.4), the pooled livers were homogenized with
an ultrasonic homogenizer (SONIFIER250, Branson, MO, USA), and the protein content
was measured according to the bicinchoninic acid method [12]. The pooled MLH wasstored at −80 ◦ C.
For analysis of NADPH-mediated metabolism of 125 I-ODV, NADPH was used as
an energy source of CYP-mediated metabolism. Adding an NADPH-generating systemproduced a state in which CYP was active. NADPH-mediated metabolism of 125 I-ODV was
examined in a mixture consisting of an NADPH-generating system (0.5 mM β-NADP+ ,5 mM MgCl2 , 5 mM G6P, and 1 U/mL G6PD), 100 mM sodium potassium phosphate buffer(pH 7.4), 50 µM EDTA disodium salt, and 1000 µg protein/20 µL pooled MLH in a final
volume of 250 µL (NADPH [+]). A mixture without the NADPH-generating system wasused as the control (NADPH [−]) to confirm NADPH dependence. The samples wereincubated at 37 ◦ C for 5, 15, 30, and 60 min with gentle shaking, ethanol was added to stop
the reactions, and the samples were centrifuged at 18,000 g for 5 min. The supernatants inInt. J. Mol. Sci. 2022, 23, 11458

11 of 13each sample were analyzed by HPLC. The mobile phase consisted of 70% 20 mM KH2 PO4
buffer (pH 4.0) and 30% acetonitrile at a flow rate of 1.0 mL/min (n = 3).
For inhibition analysis of specific isozymes of CYP-mediated metabolism of 125 I-ODV,
selective inhibitors of CYP isozymes employed in this study included α-naphthoflavone(an inhibitor of CYP1A1 and 1A2) [13], sulfaphenazole (CYP2C9) [13,14], fluconazole(CYP2C19) [15], paroxetine (CYP2D6) [13,16–19], 4-methylpyrazole (CYP2E1) [14], andketoconazole (CYP3A4) [13,14,19]. These selective inhibitors were used to elucidate the
CYP responsible for the biotransformation of 125 I-ODV. Each inhibitor was dissolved at
10 µM (dimethyl sulfoxide 0.8% v/v final concentration). The inhibition of metabolismof 125 I-ODV was examined in a mixture consisting of 50 µL of the NADPH-generating
system (NADPH [+]), 100 mM sodium potassium phosphate buffer (pH 7.4), 50 µM EDTA
disodium salt, 1000 µg protein/20 µL pooled MLH, and 25 µL inhibitor in a final volume of
250 µL. The sample was incubated at 37 ◦ C for 15 min with gentle shaking. The reaction was
stopped by adding 100 µL ethanol, and the sample was centrifuged for 5 min at 18,000 g.
The final supernatant was analyzed by HPLC using 65% 20 mM KH2 PO4 buffer (pH 4.0)
and 35% acetonitrile at a flow rate of 1.0 mL/min (n = 3).
4.4. Whole-Body Imaging of 123 I-ODV in Normal Mice
123 I-ODV was labeled and purified by the same method as 125 I-ODV. For whole-body

SPECT imaging in normal mice, all acquisitions were performed using a U-SPECT-II/CT
system (MILabs, Utrecht, The Netherlands). Three normal mice were injected with 14.5 MBq
of 123 I-ODV via the tail vein and whole-body SPECT images were acquired at 10 min/frame
immediately after injection until 60 min under 1.5% isoflurane anesthesia. The images werereconstructed using the filter-backed projection method with 16 subsets and 6 iterations.
The voxel size was set to 0.8 mm × 0.8 mm × 0.8 mm. Neither attenuation nor scattercorrection was performed. Post-reconstruction smoothing filtering was applied using a
Gaussian smooth 3D filter of 1.0 mm. Image display was performed using PMOD (ver. 3.7).
On SPECT images, the volume of interest (VOI) was drawn around the liver and gallbladder,and each percent injected dose (%ID) was calculated over time.
4.5. Metabolism of 125 I-ODV in Bile of Normal and CYP-Inhibited Mice
125 I-ODV was prepared to a radioactive concentration of 30 MBq/mL by the additionof saline. For metabolite analysis in CYP-inhibited mice, we produced model mice with reduced CYP3A4 or CYP2D6 activity by selective inhibition using ketoconazole or paroxetine.
CYP3A4-inhibited mice were injected intraperitoneally with 50 mg/kg of ketoconazole [20]at 60 min before injection of 125 I-ODV, and CYP2D6-inhibited mice were injected intraperitoneally with 30 mg/kg of paroxetine [21] at 60 min before injection of 125 I-ODV. Eachof three mice was administered 125 I-ODV via the tail vein (3.0 MBq/100 µL/mouse). At
30 min, the three fasted mice were euthanized under isoflurane, and each gallbladder wascollected. The radioactive products in bile were analyzed by TLC using methanol/acetate
at a ratio of 100:1. Bile was directly spotted onto a TLC plate.
4.6. Biological Distribution of 125 I-ODV in Normal and CYP-Inhibited Mice
For biological distribution in normal mice, 125 I-ODV was prepared to a radioactivity
concentration of 185 kBq/mL by adding saline. A total of 20 mice fasted for 6 hr wereadministered 125 I-ODV via the tail vein (18.5 kBq/100 µL/mouse). After 5, 15, 30, and60 min, normal mice were euthanized under isoflurane anesthesia (n = 4), and the following
tissues were collected: blood, brain, heart, lung, thyroid, gallbladder, liver, spleen, pancreas, stomach, small intestines, large intestines, and kidney. Tissues were weighed, andradioactivity was quantified using a γ-ray counter to calculate the %ID or percent injected
dose per gram of tissue (%ID/g).
To examine the biological distribution in CYP-inhibited mice, we produced model
mice in the same way as for the analysis of metabolism in bile. At 5, 15, 30, and 60 min afterInt. J. Mol. Sci. 2022, 23, 11458

12 of 13125 I-ODV injection, mice were euthanized under isoflurane (n = 4), and the same tissues

were collected and quantified.
4.7. Statistical Analysis
All results represent the average of least three experiments and are expressed as themean ± standard deviation. Data were analyzed using the F-test and Student’s t test, andp < 0.01 or 0.05 was considered statistically significant.
5. Conclusions
123/125 I-ODV has the potential to direct, comprehensive detection and measurementof hepatic CYP3A4 and CYP2D6 activity by a simple and less invasive approach. Imaging
and quantifying the accumulation of radioactive metabolites in excretory organs will aid
in determining the dosages of various drugs metabolized by CYP3A4 and CYP2D6 forindividualized medicine.
Author Contributions: Conceptualization, A.M. and M.K. (Masato Kobayashi), K.K.; methodology,
K.N. and K.K.; investigation, A.M., R.A. and Y.M. (Yuka Muranaka); resources, M.K. (Masanori
Kitamura) and M.K. (Munetaka Kunishima); data curation, C.S. and Y.M. (Yasuhiro Magata); writing—original draft preparation, A.M.; writing—review and editing, M.K. (Masato Kobayashi), R.N., N.S.
and K.K.; visualization, A.M.; supervision, Y.M. (Yasuhiro Magata), Y.I., M.K. (Munetaka Kunishima),
K.K.; project administration, K.K.; funding acquisition, A.M. and K.K. All authors have read andagreed to the published version of the manuscript.
Funding: This study was funded in part by Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (No. 18K19508 and 19K17194).
Institutional Review Board Statement: The animal study protocol was approved by the ethicalstandards of Kanazawa University (Animal Care Committee of Kanazawa University, AP-173851,
27 April 2021.)
Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this study
are available within the article.
Acknowledgments: We thank Mikie Ohtaki and the other laboratory staff of Kanazawa University.
Conflicts of Interest: The authors declare no conflict of interest.

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